Summary: By combining the use of a monoclonal virus derived from the HCV full length infectious rRNA with the chimpanzee model, we are making a detailed study of the immune responses following hepatitis C virus infections without the complicationg issues of quasispecies. Both T cell and B cell immune responses to HCV are being investigated using primarily the chimpanzee model, inbred mice and transgenic mice. T cell studies are continuing in an effort to identify both cytotoxic T cell and helper T cell epitopes. We are studying the proliferative and CTL responses of in chimpanzees infected by a monoclonal virus without detectable quasispecies diversity. This virus was derived from the full length, infectious cDNA clone of the HCV genome. We have inoculated a group of chimpanzees with this virus and are monitoring their immune responses in detail using a perfectly matched set to reagents. Animals that clear the infection can be reinfected with the same monotypic virus but these animals all clear the infection again. Therefore, the immune response that was sufficient to resolve the infection initially was not suffficient to completely prevent reinfection. We have used two types of vaccines to augment the natural immune responses in these recovered chimpanzees. As the recovered chimps do not make detectable antibody to the envelope glycoproteins, E1 and E2, we have vaccinated against those proteins in an attempt to induce sterilizing immunity. As resolution of HCV infections is generally associated with early, strong and diverse T cell responses, we augmented those responses in a resolved chimp by vaccinating with NS3. The E1/E2 vaccinated chimp developed high sustained levels of anti E1/E2 and the NS3 vaccinated chimp developed proliferative and cytotoxic T cell responses and upon challenge, these chimps were infected but for a shorter period of time and with a lower viral load compared to a naive vaccinated animal or a recovered, non-vaccinated chimp. We are studying ways to enhance T cell epitopes in mouse models. Previously identified Conserved CTL epitopes in the HCV core protein have been mutated to look for cross reacting epitopes that actually function better than the wild type. Both in bred and HLA A2.1 transgenic mice have been used in these experiments. It has long been known that chimpanzees can be reinfected with HCV leading to the suggestion that natural infection does not elicit a protective immune response. We wished to compare the dynamics of infection and in vivo responses in previously infected chimpanzees versus naive chimpanzees upon challenge/rechallenge with a monotypic HCV. Serum was taken from a chimpanzee infected by intrahepatic inoculation of RNA transcribed in vitro from a cDNA clone. Naive (n=1) and recovered chimpanzees (n=2) were challenged with the serum and tested for evidence of hepatitis, viremia and for changes in intrahepatic cytokine levels. At the time of the challenge the recovered chimps were EIA 3.0 positive but had no anti-E1E2 antibody, none of the chimps had a preexisting HCV specific, proliferative T cell response in the blood. All animals became infected with the monotypic virus. The naive chimpanzee, Ch1605, developed a typical acute infection: increasing viremia from 1 wk post inoculation (p.i.) (peak at wk 7, 1x10E6 RNA copies/ml) and ALT elevations (peak at wk 9, 7x over base line). This animal eventually cleared the infection after approximately one year. The recovered chimpanzees X0186 and 1552 had viremia for only 4 wks at significantly reduced titers (< 2x10E3 RNA copies/ml) and only minor ALT elevations. At the time HCV RNA was detected, both displayed a proliferative, HCV-specific T cell response that was significantly stronger for ChX0186 than for Ch1552. Intrahepatic levels of IFN-gamma, TNF-alpha, and IL4 were measured for Ch1605 and Ch1552. In Ch1605 only IFN-gamma levels increased at 10 wks p.i. (8x above base line). This increase coincided with the ALT elevation. In Ch1552 IFN-gamma and TNF-alpha levels increased. This increase at 2 weeks p.i., and reached levels 30x (IFN-gamma) and 9x (TNF-alpha) above base line. These increases also coincided with the brief, low ALT elevation observed in this animal. Chimpanzees that have cleared infections are reinfectable with a monotypic virus encoding exactly the same sequence or a subset of the original infecting virus. However, these animals exhibit significantly reduced viremia, minor ALT elevations and clear the virus within a relatively short period of time. This data indicates that there is a memory immune response induced during the primary infection that controls the secondary infection. This immune response appears to be T cell rather than antibody based and is targeted to the liver. These results have promising implications for the development of HCV vaccines.